Integrins are heterodimers composed of non-covalently associated α- and β-chains which connect cells to the extracellular matrix or to other cells (1). In addition to acting as mechanical links between the cytoskeleton and extracellular ligands, integrins are signal transducing receptors which influence processes such as cell proliferation, cell migration and cell differentiation (2-4). Integrins can be grouped into subfamilies based on shared β-chains, shared ligand binding properties, or shared structural features of the α-chains. Currently 17 α-chains and 8 β-chains have been identified (5). Of the subfamilies with shared β-chains, the Pβ1 subfamily has the most members. To date, 11 integrin α-chains associated with the β1-chain have been identified and characterized, α1-α10 and αv (5).
Several integrins bind the sequence RGD in their respective ligands (1). Of those integrins identified so far, α4-, α5-, α8-, αIIb- and αv-chains form heterodimers that mediate RGD-dependent interactions. The ligands containing RGD are generally found in the interstitial type of extracellular matrix. Major non-RGD dependent ligands include various collagen and laminin isoforms. Although both collagens and laminins contain the RGD sequence in their primary sequences, these RGD sequences are cryptic (6-9) and normally not accessible to cells in the native proteins, but they may be exposed during growth and reorganization events of the extracellular matrix.
Another subdivision of integrins can be made based on structural similarities of the α-chains. A number of integrins contain an extracellular I-domain (10, 11) which is homologous to collagen binding A-domains present in von Willebrand factor (12). The I-domain constitutes an inserted domain of approximately 200 amino acids which is present in 8 known integrins (α1, α2, α10, αL, αM, αX, αD and αE) (5, 10). Structural analysis of integrin I-domains crystallized in the presence of Mg2+ have revealed the presence of a characteristic “MIDAS” (metal ion dependent adhesion site) motif, shown to be critical for ligand binding (13). Integrin α-chains containing the I-domain are not cleaved into heavy and light chains, although the rat α1 chain possesses a proteolytic cleavage site near the membrane spanning region (14, 15). For I-domain integrins the principal ligand binding sites are found within the I-domain (10). Known ligands for I-domains found within the β1 integrin subfamily include laminins and collagens (α1β1 and α2β1 integrins) (16-19), and Echovirus (α2β1 integrin) (20).
Structure comparisons have suggested that integrins fold into a so-called 7-bladed β-propeller structure which forms one globular domain with the ligand binding region on the upper surface (21). The I-domain is inserted between blade 2 and 3 in this propeller and divalent cation binding sites are located on the lower surface in blades 4-7 (22, 23). Studies of β2 integrins have revealed that proper folding of the β2-chain is dependent on the presence of the αL-chain but that the I-domain folds independently of other structural elements in the α- and β-chains (24). In integrin α-chains, a less conserved stalk region separates the predicted β-propeller from the short transmembrane region. This stalk region is possibly involved in transducing conformational changes between the extracellular and intracellular regions, as well as mediating protein-protein interactions. Although integrins take part in cell signalling events, the cytoplasmic tail is short and lacks enzymatic activity. The sequence GFFKR is conserved in a majority of integrin α-subunits cytoplasmic tails and has been shown to be important for calreticulin binding (25).
Cellular interactions with the extracellular matrix during muscle formation and in muscular dystrophy have received increased interest during the past years. In the early 1960's a mutant was described in Drosophila which was characterized by the detachment of muscles from their attachment points at the time of the first embryonic muscle contraction, causing the embryos to assume a spheroid shape (26). The mapping of the molecular defect in the lethal myospheroid mutant in 1988 to an integrin β-chain (27), was the first evidence for a role of integrins in maintaining muscle integrity. More recently, refined analysis of Drosophila mutants have indicated distinct roles for integrins in muscle endpoint attachments and sarcomere structure (28). The Drosophila integrins are all cleaved α-chains and share many features with vertebrate integrins such as the ability to cluster into focal contacts (29).
The finding that inactivation of the α7 integrin gene in mouse (30), as well as mutations in the human ITGA7 gene (31), both cause muscular dystrophy affecting mainly muscle attachment points, indicates a striking conservation of integrin function during evolution. Of the 11 members of the β1 subfamily, α7 exists as a major integrin α-chain (32, 33) associated with the β1D integrin chain in the adult skeletal muscle sarcolemma (34). Intriguingly, mutations in the basement membrane protein laminin α2-chain (35-37) cause a more severe disease than that observed for the laminin receptor integrin α7β1 (30). This indicates that other receptors for laminins exist in muscle.
A novel integrin has recently been identified on cultured human fetal muscle cells (38). The present invention is related to, inter alia, the cloning and characterization of this novel I-domain containing, β1-associated integrin chain, which is expressed in muscle tissues.